Objective: To determine genotype, nucleotide sequence homology and phylogenesis of Orientia tsutsugamushi isolated from Shandong, China.
Methods: Orientia tsutsugamushi isolated from patients, Apodemus agrarius and Leptotrombidium scutellare in Shandong area were identified by nested-PCR. On the basis of the nucleotide sequence of the gene that encoding the Ot M, 56 x 10(3) antigen, the primers were frequently used in Japan and Korea. Nucleotide sequences of three isolates were determined. The DNA sequences were compared with nucleotide sequences of Orientia tsutsugamushi registered in GenBank for sequence homology analysis. Phylogenetic analysis of the isolates and'some published sequences was carried out with Neighbor-joining method by MEGA 3.1 software.
Results: 481- 507 bp DNA fragments encoding Orientia tsutsugamushi M, 56 x 10(3) protein were amplified successfully in the samples of Gilliam, Karp, Kato and Shandong isolates by group-specific primers. The corresponding target fragments of the three international reference strains of Gilliam, Karp, and Kato were amplified successfully with each of their own type specific primers. 523 bp DNA fragments were amplified successfully from Shandong isolates by the nPCR with Kawasaki-specific primer, and no DNA fragment was amplified by the nPCR with Gilliam, Karp, Kato, Kuroki and Saitama-specific primer. Comparing with the sequences of Orientia tsutsugamushi registered in GenBank, all the Shandong isolates shared higher than 95% nucleotide sequence homology with Kawasaki strain founded in Japan. Data from phylogenetic analysis showed that Shandong isolates belonged to the same branch with Kawasaki strain.
Conclusion: To facilitate international comparison and communication, the primers should be employed in the Orientia tsutsugamushi research in China. Orientia tsutsugamushi isolated in China were similar to Kawasaki strain